Development and Evaluation of an XTT Assay for Rapid Susceptibility Testing of Aspergillus Spp.

C. ANTACHOPOULOS, J. MELETIADIS, T. SEIN, E. ROILIDES, T. J. WALSH;

Author address: 

Natl. Cancer Inst., Bethesda, MD.

Abstract: 

Background: Invasive aspergillosis in immunocompromised hosts requires prompt treatment with appropriate antifungal agents. We developed an XTT colorimetric assay of metabolic activity for rapid susceptibility testing of Aspergillus spp. Methods: Susceptibility to amphotericin B (AMB) and voriconazole (VRC) was tested for 39 clinical isolates of Aspergillus spp (16 A. fumigatus, 11 A. flavus, 12 A. terreus). At 6 and 8 h after inoculation for A. fumigatus and A. flavus and 12 h after inoculation for A. terreus, 100 μg/ml XTT and 25 μM menadione were added and absorbance measured at 450 nm after incubation at 37oC for 2 h. 106 conidia/ml were used for A. fumigatus and A. terreus and 105 conidia/ml for A. flavus, as lower inocula resulted in very low XTT conversion at these timepoints. Data were analyzed with the sigmoid Emax model and compared with visual and spectrophotometric determinations of MIC-0 (lowest drug concentration showing no growth) at 48 h according to CLSI M38-A method. Results: The model fit well the XTT and 48h spectrophotometric readings (median r2: 0.97 and 0.93 respectively); for A. fumigatus (both drugs) and A. terreus (AMB) the XTT dose response curves were shifted to the left by one to two dilutions compared to those of 48 h spectrophotometric readings. For AMB, the CLSI MIC-0 at 48 h corresponded to the lowest drug concentration showing metabolic activity 8804; 10% of drug-free control for A. fumigatus (6 h), 35% for A. flavus (6 h) and 10% for A. terreus (12 h). For VRC MIC-0, the corresponding metabolic activity for these species and timepoints was 25%, 40% and 40% respectively. By using these cutoff levels for determination of MIC-0 with the XTT assay and adjusting for the shifting of curves, the relative agreement (± 1 dilution) between the early XTT and CLSI determination of MIC-0 of AMB and VRC ranged from 91% to 100% for all species and timepoints. Conclusions: The XTT assay can be used for rapid susceptibility testing of Aspergillus spp, yielding results that are in agreement with the CLSI method.
2006

abstract No: 

M-1566

Full conference title: 

46th Interscience Conference on Antimicrobial Agents and Chemotherapy
    • ICAAC 46th